The means used to deliver medically useful substances can significantly affect their efficacy. The standard route of administration for many such substances is either oral, intravenous, or subcutaneous. Each has inherent limitations which can affect the therapeutic utility of the substances being delivered. Furthermore, many protein-based drugs have short half-lives and low bioavailabilities, factors that must be considered in their formulation and delivery. Although various devices have been developed to deliver medically useful substances, including portable pumps and catheters, there is still a significant need for improved delivery devices.
Many medically useful substances, including proteins, glycoproteins, and some peptide and nonpeptide hormones, are more efficiently produced by cultured cells than via artificial synthetic routes. Appropriate cells are typically cultured in bioreactors, and the desired product purified therefrom for administration to the patient by standard means, e.g. orally or by intravenous or subcutaneous injection. Alternatively, the cells may be implanted directly into the patient, where they produce,and deliver the desired product (see, e.g., U.S. application Ser. Nos. 07/787,840 and 07/789,188). While this method has a number of theoretical advantages over injection of the product itself, including the possibility that normal cellular feedback mechanisms may be harnessed to allow the delivery of physiologically appropriate levels of the product, it introduces additional complexities. One of these concerns the appropriate environment for the cells at the time of implantation. It would be desirable to organize the cells of the implant in a form that is compatible with the natural in vivo environment of the cell type comprising the implant (fibroblasts, for example, exist naturally in a rich network of extracellular matrix composed primarily of collagen). There is also a need in some cases to ensure that the implanted cells remain localized to a defined site in the patient""s body, so that they can be monitored and perhaps removed when no longer needed.
One technique that has been tested for this purpose utilizes an implantation device consisting of a solid, unitary piece of collagen gel (a xe2x80x9ccollagen matrixxe2x80x9d) in which the cells are embedded (e.g., Bell, U.S. Pat. No. 4,485,096). Other substances, such as polytetrafluoro-ethylene (PTFE) fibers (Moullier et al., Nature Genetics, 4:154, 1993; WO 94/24298), may be included in the collagen implant to impart strength or other desirable characteristics to the collagen gel.
It has been found that the function of collagen matrices can be substantially improved by the addition of microspheres (i.e., microcarriers of any shape) to the collagen matrix, thereby forming what is herein termed a xe2x80x9chybrid matrixxe2x80x9d. This may be accomplished by mixing microspheres with the cells and soluble collagen prior to gelling of the collagen to form the matrix. If desired, the microspheres and cells can be cultured together for a period which permits the cells to adhere to the microspheres before addition of the non-gelled collagen solution; alternatively, the three constituents can be mixed essentially simultaneously or in any desired order, followed by gelation of the soluble collagen within the mixture, to form a gelled mixture consisting of insoluble collagen fibrils, cells and microspheres. This gelled mixture gradually becomes smaller through the exclusion of liquid to form a solid, relatively resilient, implantable unit that contains both the microspheres and the cells embedded in the insoluble collagen fibril network. When the microspheres are also composed largely of collagen, the resulting matrix is herein termed a xe2x80x9chybrid collagen matrixxe2x80x9d. It is understood that the microspheres in the hybrid collagen matrices could contain substances in addition to collagen.
The invention thus includes an article, composition, or device having a body made of matrix material that includes insoluble collagen fibrils, and disposed within the body:
(a) a plurality of vertebrate cells (particularly mammalian cells such as cells derived from a human, chimpanzee, mouse, rat, hamster, guinea pig, rabbit, cow, horse, pig, goat, sheep, dog, or cat); and
(b) a plurality of microspheres, each of which preferably consists primarily of (i.e., greater than 50% of its dry weight is) one or more substances selected from a list including collagen (preferably type I collagen), polystyrene, dextran, polyacrylamide, cellulose, calcium alginate, latex, polysulfone, glass (e.g., glass coated with a gel such as collagen, to improve adherence of cells), and gelatin (e.g., porous gelatin). Generally at least 70%, and preferably at least 80% (most preferably between approximately 90% and approximately 100%, e.g., at least 95%) of each microsphere""s dry weight is one or more of the listed substances. Commercial examples of microspheres which are described as consisting essentially of purified collagen include ICN Cellagen(trademark) Beads and Cellex Biosciences macroporous microspheres. The microspheres are preferably of a porous consistency, but may be smooth, and typically have an approximately spherical shape with a diameter of approximately 0.1 to 2 mm (e.g., between approximately 0.3 and 1 mm). Of course, the shape and size of microspheres from any particular lot or preparation will vary within manufacturing tolerances.
The article may be configured to be implanted into an animal, e.g., a mammal such as a human patient, or may be designed for producing cellular products in vitro; e.g., in an extracorporeal bioreactor apparatus having a means for shunting blood from an animal to the article and then back into a blood vessel of the animal, or in a bioreactor or other vessel from which medium containing the desired cellular product can be recovered for purification and the preparation of a pharmaceutical agent.
The cells may be derived from one or more cells removed from the patient, and preferably are genetically engineered (e.g., transfected) cells containing exogenous DNA encoding one or more medically useful polypeptides such as an enzyme, hormone, cytokine, colony stimulating factor, angiogenesis factor, vaccine antigen, antibody, clotting factor, regulatory protein, transcription factor, receptor, or structural protein. Examples of such polypeptides include human growth hormone (hGH), Factor VIII, Factor IX, erythropoietin (EPO), albumin, hemoglobin, alpha-1 antitrypsin, calcitonin, glucocerebrosidase, low density lipoprotein (LDL) receptor, IL-2 receptor, globins, immunoglobulins, catalytic antibodies, the interleukins, insulin, insulin-like growth factor 1 (IGF-1), insulinotropin, parathyroid hormone (PTH), leptin, an interferon (IFN) (e.g., IFNxcex1, IFNxcex2, or IFN-xcex3), nerve growth factors, basic fibroblast growth factor (bFGF), acidic FGF (aFGF), epidermal growth factor (EGF), endothelial cell growth factor, platelet derived growth factor (PDGF), transforming growth factors, endothelial cell stimulating angiogenesis factor (ESAF), angiogenin, tissue plasminogen activator (t-PA), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), follicle stimulating hormone (FSH), xcex1-galactosidase, xcex2-gluceramidase, xcex1-iduronidase, xcex1-L-iduronidase, glucosamine-N-sulfatase, xcex1-N-acetylglucosaminidase, acetylcoenzyme A:xcex1-glucosaminide-N-acetyltransferase, N-acetylglucosamine-6-sulfatase, xcex2-galactosidase, N-acetylgalactosamine-6-sulfatase, and xcex2-glucuronidase. Alternatively, the exogenous DNA can contain a regulatory sequence, and optionally other elements, that will activate expression of an endogenous gene (for example, using homologous recombination as described in WO94/12650-PCT/US93/11704, which is incorporated by reference herein).
Generally any type of cell which is capable of attaching to collagen and/or the microspheres, and which exhibits a desirable property such as expression of a medically useful cellular product or performance of an essential structural or metabolic function, can be utilized in the matrices of the invention. Examples include adipocytes, astrocytes, cardiac muscle cells, chondrocytes, endothelial cells, epithelial cells, fibroblasts, gangliocytes, glandular cells, glial cells, hematopoietic cells, hepatocytes, keratinocytes, myoblasts, neural cells, osteoblasts, pancreatic beta cells, renal cells, smooth muscle cells and striated muscle cells, as well as precursors of any of the above. If desired, more than one type of cell can be included in a given matrix. The cells may be present as clonal or heterogenous populations.
The collagen in the matrix material is preferably type I, but may be any other type of collagen. The matrix material may optionally include two or more types of collagen (e.g., selected from types I, II, III, IV, V, VI, VII, VIII, IX, X, and XI), as well as any additional components that impart desirable characteristics to the resulting matrix: e.g., agarose, alginate, fibronectin, laminin, hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin sulfate, sulfated proteoglycans, fibrin, elastin, tenascin, heparin or polysaccharides such as cellulose, starch or dextran. Any of the above mentioned collagenous and non-collagenous components may be derived from human sources or from another animal source. One could also include collagen or non-collagen fibers disposed within the device. Collagen fibers can be in the form of cross-linked collagen threads dispersed within the body of the matrix material. Non-collagen fibers can, for example, be made of a material that includes nylon, dacron, polytetrafluoro-ethylene, polyglycolic acid, polylactic/polyglycolic acid polymer mixtures, polystyrene, polyvinylchloride co-polymer, cat gut, cotton, linen, polyester, or silk.
Large numbers of cells can be contained within the hybrid matrices. For example, hybrid matrices can be prepared which contain at least approximately two (and preferably approximately three) times as many cells as matrices prepared with soluble collagen alone, assuming the number of cells inoculated and the initial production volume are equivalent. The total amount of polypeptide expressed by the cells embedded in a given hybrid matrix in a given time period is typically significantly higher (e.g., at least 50% higher, preferably at least 100% higher, and more preferably at least 200% higher) than achieved with a standard collagen matrix prepared from an equivalent volume of starting material.
Any of the above-described hybrid matrices of the invention can also contain one or more (e.g., at least 2, 3, 4, 5, 6, 8, or 10) agents intended to improve the functioning of the matrix, e.g., by increasing proliferation and/or maintenance of the cells. These agents can include, for example, factors which promote vascularization, cytokines, or growth factors. While the agent used in a particular hybrid matrix and the polypeptide, e.g., a medically useful polypeptide, produced by the cells in the matrix can be the same substance, the two entities will generally be different. The agent can be added directly to the mixture used to make the hybrid matrices or can be bound to or encapsulated within a solid substrate which is added to the same mixture. The solid substrate can be the microspheres themselves or can be a separate entity or entities (e.g., multiple particles of the solid substrate, or a single piece, embedded in the matrix. The solid substrate can have heparin or heparan sulfate proteoglycan bound to it, as a means for promoting binding of the agent. An example of such a solid substrate is one that consists primarily of agarose (e.g., Sepharose(trademark), Affi-Gel(trademark) Heparin Gel, or Heparin-agarose), with or without heparin or heparan sulfate proteoglycan bound to it; such a solid substrate can also contain calcium alginate. Other substances from which the solid substrates can be manufactured include collagen, gelatin, ethylene-vinyl acetate, polylactide/glycolic acid co-polymer, fibrin, sucrose octasulfate, dextran, polyethylene glycol, an alginate, polyacrylamide, cellulose, latex, polyhydroxyethylmethacrylate, nylon, dacron, polytetrafluoro-ethylene, polyglycolic acid, polylactic acid, polystyrene, polyvinylchloride co-polymer, cat gut, cotton, linen, polyester, and silk. The solid substrate can be in a variety of physical forms, e.g., beads, irregular particles, sheets, or threads. When the agent is encapsulated in the solid substrate, the agent is released gradually over time, e.g., due to enzymes that act on the solid substrate.
Examples of agents which can be used in the matrices include basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), VEGF-A, VEGF-B, VEGF-C, VEGF-D, acidic fibroblast growth factor (aFGF), endothelial cell growth factor, platelet-derived growth factor (PDGF), endothelial cell stimulating angiogenesis factor (ESAF), leukotriene C4, a prostaglandin, insulin-like growth factor 1 (IGF-1), granulocyte colony stimulating factor (G-CSF), angiogenin, transforming growth factor-xcex1 (TGF-xcex1), transforming growth factor-xcex2 (TGF-xcex2), ascorbic acid, epidermal growth factor (EGF), or oncostatin M.
The bioactive concentration of each agent will vary greatly. A starting range is provided by the manufacturer and is usually based on a standard bioactivity assay using, for example, degree of cell proliferation as the end point. Typically, the agent is bound at a broad range of concentrations (lowest being what is reported as bioactive by the vendor, highest being as much as 1000xc3x97 that of the reported concentration) to a substrate such as heparin-Sepharose beads; the beads are incorporated into an HCM; and the release of the agent over time in vitro is monitored using an appropriate detection system (e.g., an immunoassay). For these release assays, the matrices are placed in growth medium containing 10% serum. Once it is determined that the matrices release detectable amounts of the agent, a bioactivity assay is performed. Matrices containing a range of agent concentrations can, for example, be placed on porous inserts (3 to 8 xcexcm pores) above cells that are known to proliferate in response to the agent (e.g, endothelial cells for VEGF and bFGF, fibroblasts for bFGF and PDGF, as indicated by manufacturer), and cell growth curves determined. The results of the in vitro bioactivity assay are evaluated and doses that are not deemed bioactive as well as doses that are determined to be xe2x80x9ctoxicxe2x80x9d (i.e., lead to cell numbers lower than control) are noted. In order to determine the optimal concentration of agent per matrix, matrices containing the agent at a range of concentrations based on the in vitro bioactivity results are implanted in immunocompromised mice. The optimal agent concentration per matrix is typically the concentration that allows for the maximum amount of therapeutic protein to be released for the maximum amount of time in vivo.
Instead of (or in addition to) the described agent per se being dispersed in the body of the matrix material, the hybrid matrices can contain, in addition to the first population of cultured vertebrate cells genetically engineered to express a polypeptide (e.g., a medically useful polypeptide), a second population of cultured vertebrate cells expressing and secreting one or more (e.g., at least 2, 3, 4, 6, 8, or 10) of the agents. The cultured vertebrate cells of the second population can be genetically engineered (as described below) to express the agent, or can be a cell which produces the agent without the benefit of genetic engineering. In the latter case, if the cell does not constitutively produce the polypeptide, or produces it in very low amounts, the cell can be induced to produce the agent, or produce it higher amounts by gene activation. The first and second populations can be the same population of cells transfected with a DNA encoding the polypeptide and a DNA encoding the agent (or encoding the enzyme(s) necessary to produce the agent, such as for ascorbic acid); alternatively, the cells can be transfected with a single DNA encoding both the polypeptide and the agent or agent-producing enzyme(s). The matrices of the invention can contain additional populations of cultured vertebrate cells which express and secrete additional examples of the above described agents. The cultured vertebrate cells of the agent-producing populations can be adipocytes, astrocytes, cardiac muscle cells, chondrocytes, endothelial cells, epithelial cells, fibroblasts, gangliocytes, glandular cells, glial cells, hematopoietic cells, hepatocytes, keratinocytes, myoblasts, neural cells, osteoblasts, pancreatic beta cells, renal cells, smooth muscle cells, striated muscle cells, or precursors of any of the above. The cells are preferably human cells, but can be cells of any vertebrate (e.g., a mammal such as a non-human primate, pig, cow, horse, goat, sheep, dog, cat, mouse, rat, rabbit, guinea pig or hamster). When cells producing the agent are included in the matrix, one can optionally include the solid substrate as well, to bind a portion of the agent as it is secreted from the cells. This provides a means for controlling the rate of release of the agent from the hybrid matrix.
In one preferred embodiment, the hybrid matrices of the invention contain keratinocytes, e.g., (a) as the cells which produce the polypeptide, (b) as the cells which produce the agent, (c) both (a) and (b), or (d) as a population which produces neither the polypeptide nor the agent. Hybrid matrices to which the keratinocytes are added are preferably those containing fibroblasts as cells producing the above-described polypeptide (e.g., a medically useful polypeptide) and/or agent. The keratinocytes and the fibroblasts can be obtained from the same individual, and one or more keratinocyte differentiation factors (e.g., calcium ions at a concentration of 1.5-2 mM, TGF-xcex2, or keratinocyte differentiation factor-1 (KDF-1)) can be added to the hybrid matrix.
In a similar manner, the hybrid matrices of the invention may contain endothelial cells, preferably in addition to fibroblasts producing a medically useful polypeptide, and even in addition to both fibroblasts and keratinocytes. The endothelial cells and fibroblasts can be obtained from the same individual. One or more endothelial differentiation factors (e.g. vascular endothelial growth factor or basic fibroblast growth factor at 10 ng-10 xcexcg) can be added to the hybrid matrix to induce the formation of endothelial tubes within the matrix. The growth factors can be added directly to the matrix during formation, or added to the matrix growth medium, or both.
The hybrid matrix of the invention is generally prepared by a process that includes the following steps:
forming a mixture that includes (a) a plurality of vertebrate cells; (b) a plurality of microspheres, each of which preferably consists primarily of one or more substances selected from the list consisting of collagen, polystyrene, dextran, polyacrylamide, cellulose, calcium alginate, latex, polysulfone, and glass; and (c) a solution comprising soluble collagen;
causing the soluble collagen in the mixture to form a gel of insoluble collagen fibrils in which the cells and the microspheres are embedded; and
exposing the gel to culture conditions which cause the gel to become smaller by the exclusion of liquid, thereby forming the body of the article. Gelation is typically triggered by raising the pH of the relatively acidic collagen solution to above 5, e.g., by addition of concentrated, buffered culture medium, whereupon the collagen forms insoluble fibrils. When this step is carried out in a mold, the gel will take the shape of the interior of the mold. Generally the contraction of the gel is effected by the cells in the mixture, which attach to the fibrils and cause the gel to contract to a smaller version of the molded shape (e.g., a disk, as in the case where the mold is a petri dish which is cylindrical in shape). The matrix may be utilized immediately after manufacture, may be cultured to increase the number of cells present in the matrix or to improve their functioning, or may be cryopreserved indefinitely at a temperature below 0xc2x0 C. They can also be stored temporarily in a higher temperature refrigerator at, for example, about 4xc2x0 C.
For making those hybrid matrices containing one or more of the above-described agents, the relevant agents (free or bound to any of the above-described solid substrates) are added, together with the other components listed above, to the mixture. The agent and solid substrate can be added to the mixture together or separately, in any order. Additionally or alternatively, the mixture can contain a second (and, optionally, a third, fourth, fifth, sixth, or more) population of cultured vertebrate cells secreting one or more of the described agents. The mixture can also contain one or more of the above-described solid substrates that include one or more substances (e.g., heparin or heparan sulfate) which binds to an agent. For making hybrid matrices containing keratinocytes, the keratinocytes can be added to the mixture prior to the contraction step, or they can be added to the body of the composition after the gel has contracted.
In making any of the hybrid matrices of the invention, the gel can be formed in a flat-bottomed mold filled with the mixture to a depth of about 0.18 cm (e.g., about 0.1 cm to about 0.3 cm, preferably about 0.15 cm to about 0.21 cm). For example, the gel can be formed in a flat-bottomed cylindrical mold having an internal radius (r) using a mixture having a volume (V), such that r2/V is about 1.8 (e.g., 1.5 to 2.0). The invention includes hybrid matrices resulting from use of the specified depth of mixture, and/or the specified ratio of r2/V, and thus having a characteristic thickness. The gel can be formed, for example, in a cylindrical mold having a radius of about 2.65 cm, using a volume of 4 ml, or in a mold the radius of which is other than 2.65 cm (i.e., larger or smaller), with a proportional change in volume of mixture used.
A medically useful polypeptide, such as one listed above, may be delivered to a patient by a treatment method that involves providing a hybrid matrix containing cells which secrete the polypeptide of interest, and implanting the article in the patient in a selected site, such as a subcutaneous, intraperitoneal, intraomental, sub-renal capsular, inguinal, intramuscular or intrathecal site. Where the polypeptide is one which promotes wound healing (e.g., PDGF or IGF-I), the matrix may be implanted at the site of a preexisting wound. As discussed above, the cells may be derived from one or more cells removed from the patient, and are preferably transfected in vitro with exogenous DNA encoding the polypeptide. Alternatively, they may be cells which naturally secrete the polypeptide or perform the desired metabolic function (e.g., hepatocytes or pancreatic beta cells). The cells can be induced to secrete the polypeptide, secrete higher amounts of the polypeptide, or perform the desired metabolic function by gene activation. Appropriate hybrid matrices for delivering a polypeptide to a patient can be any of those described above.
In another embodiment, the medically useful polypeptide may be administered to the patient by shunting a portion of the patient""s blood through the apparatus described above, so that the polypeptide secreted by the cells in the hybrid matrix mixes with the blood. Generally, any such apparatus known to those in that field can be adapted to accommodate the matrix of the invention. For example, blood shunted into a device which contains a perm-selective membrane surrounding a matrix of the present invention will result in the delivery of a therapeutic product of the matrix to the blood. A device similar to an artificial pancreas (Sullivan et al., Science 252:718-721, 1991) may be used for this purpose. Again, any of the hybrid matrices described herein can be used for such devices.
Yet another use for any of the hybrid matrices of the invention is as a means for producing a polypeptide in vitro. This method includes the steps of placing the hybrid matrix under conditions whereby the cells in the matrix express and secrete the polypeptide; contacting the matrix with a liquid such that the cells secrete the polypeptide into the liquid; and obtaining the polypeptide from the liquid, e.g., by standard purification techniques appropriate for the given polypeptide. In one embodiment, the matrix is anchored to a surface and is bathed by the liquid; alternatively, the matrix floats freely in the liquid. Cells embedded in the hybrid matrix function at a high level in a small space. Furthermore, the first step in purification of the expressed polypeptide (removal of the cells from the medium) is considerably more efficient with the matrices than with most standard methods of cell culture.
As used herein, a xe2x80x9csolid substratexe2x80x9d is an object, or a plurality of objects (configured, for example, as particles or threads), which acts as a reservoir or depot for a substance (e.g., a factor that promotes vascularization) that is contained within or is bound to the solid substrate. The substance is gradually released from the solid substrate into its environment. Where the solid substrate is in the form of beads, the beads have, generally, an approximately spherical shape and have a diameter of approximately 0.005-2.0 mm. Where the solid substrate is in the form of threads, the threads are generally about 0.01-1.0 mm in diameter. The threads can be folded into a meshwork or cut into small pieces (of approximately 5-10 mm) prior to gel formation. Where the threads are folded, their length should be, for example, about 1xc3x97 to 3xc3x97 (e.g., about 2xc3x97) the diameter of a mold used to produce the relevant hybrid matrix.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although other methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples described herein are illustrative only and not intended to be limiting.
Other features and advantages of the invention are apparent from the claims, and from the detailed description provided below.